Summary: The apical membranes of water-tight epithelia (e.g. mammalian collecting duct and toad urinary bladder) are nearly water-impermeable under basal conditions; the lipid composition and structure responsible for this low permeability is unknown. In response to antidiuretic hormone (ADH) subapical vesicles containing water channels (WCV) fuse with the apical membrane, resulting in marked increases in water permeability. The mechanisms underlying water channel insertion into and removal from the apical membrane remain unclear. In addition the actual structure of the ADH-induced water channel is poorly defined. The present studies will utilize newly developed methods for isolation of membrane fractions from toad urinary bladder and rat collecting duct to characterize the permeability properties of the apical membrane and WCV and relate these properties to their lipid and protein structures. Apical membranes purified by affinity labeling followed by magnetic selection will be prepared from unstimulated and ADH-stimulated bladders. Permeabilities to water, protons, urea, and small solutes will be examined and correlated with determinations of lipid and protein structure. WCV from toad bladder and rat collecting duct will be purified by flow cytometry using entrapped fluorescein dextrans. Again, permeabilities will be correlated with lipid and protein composition. These studies will characterize the mechanisms of water flow in biological membranes in the absence as well as the presence of water channels and will further define potential mechanisms by which functional water channels are inserted into and removed from the apical membrane.